Lanjuan Shen1, Cheng Ji1, Jian Lin2, Hongping Yang3. 1. Department of Orthopedics, The First People's Hospital of Hangzhou, No.261, Huansha Road, Shangcheng District, Zhejiang Province, Hangzhou, 310000, China. 2. Department of Orthopedics, Hangzhou Xiaoshan Hospital of Traditional Chinese Medicine, Zhejiang Province, Hangzhou City, China. 3. Department of Orthopedics, The First People's Hospital of Hangzhou, No.261, Huansha Road, Shangcheng District, Zhejiang Province, Hangzhou, 310000, China. Syhl202004@163.com.
Abstract
INTRODUCTION: A disintegrin and metallopeptidase with thrombospondin type 1 motif 6 (ADAMTS6)-derived circular RNA (circADAMTS6; hsa_circ_0008667) is a novel regulator in interleukin (IL)-1β-induced apoptosis of human chondrocytes (HCs). Here, we planned to probe into its role and mechanism underlying HCs injury in osteoarthritis. MATERIALS AND METHODS: Real time-quantitative PCR and immunoblotting estimated the abundance of RNA and protein, respectively. Cell proliferation and apoptosis were measured by WST-8, EdU, fluorescein isothiocyanate, and caspase3/7 activity assays. Levels of inflammatory cytokines (IL-6 and tumor necrosis factor-α), apoptosis-related proteins (Bcl-2 and Bcl-2-associated X protein), extracellular matrix (ECM)-related proteins (matrix metalloproteinase-13 and collagen type II alpha-1), and PI3K/AKT/mTOR signaling pathway-related proteins (AKT, mTOR, phosphorylated-AKT, and phosphorylated-mTOR) were evaluated by enzyme-linked immunosorbent assays and immunoblotting. Target relationship was confirmed by dual-luciferase reporter, Argonaute-2 immunoprecipitation and RNA pull-down assays. RESULTS: Abundances of circADAMTS6 and phosphoinositide-3-kinase regulatory subunit 3 (PIK3R3) were underexpressed, and microRNA (miR)-324-5p was elevated in human osteoarthritic tissues and IL-1β-induced HCs. Overexpressing circADAMTS6 and inhibiting miR-324-5p enhanced proliferation and ECM synthesis, but suppressed apoptosis and inflammatory response in IL-1β-challenged HCs. Besides, silencing circADAMTS6 caused similar effects of IL-1β stress on HCs. Mechanically, there was a direct interaction between miR-324-5p and circADAMTS6 or PIK3R3, and IL-1β-induced activation of PI3K/AKT/mTOR signaling pathway was suppressed by circADAMTS6 overexpression and miR-324-5p silencing. Furthermore, counteractive effects of miR-324-5p upregulation on circADAMTS6 overexpression and PIK3R3 knockdown on miR-324-5p silencing were observed. CONCLUSION: CircADAMTS6-miR-324-5p-PIK3R3 axis might participate in IL-1β-induced HCs dysfunction via competing endogenous RNA mechanism and the PI3K/AKT/mTOR signaling pathway.
INTRODUCTION: A disintegrin and metallopeptidase with thrombospondin type 1 motif 6 (ADAMTS6)-derived circular RNA (circADAMTS6; hsa_circ_0008667) is a novel regulator in interleukin (IL)-1β-induced apoptosis of human chondrocytes (HCs). Here, we planned to probe into its role and mechanism underlying HCs injury in osteoarthritis. MATERIALS AND METHODS: Real time-quantitative PCR and immunoblotting estimated the abundance of RNA and protein, respectively. Cell proliferation and apoptosis were measured by WST-8, EdU, fluorescein isothiocyanate, and caspase3/7 activity assays. Levels of inflammatory cytokines (IL-6 and tumor necrosis factor-α), apoptosis-related proteins (Bcl-2 and Bcl-2-associated X protein), extracellular matrix (ECM)-related proteins (matrix metalloproteinase-13 and collagen type II alpha-1), and PI3K/AKT/mTOR signaling pathway-related proteins (AKT, mTOR, phosphorylated-AKT, and phosphorylated-mTOR) were evaluated by enzyme-linked immunosorbent assays and immunoblotting. Target relationship was confirmed by dual-luciferase reporter, Argonaute-2 immunoprecipitation and RNA pull-down assays. RESULTS: Abundances of circADAMTS6 and phosphoinositide-3-kinase regulatory subunit 3 (PIK3R3) were underexpressed, and microRNA (miR)-324-5p was elevated in human osteoarthritic tissues and IL-1β-induced HCs. Overexpressing circADAMTS6 and inhibiting miR-324-5p enhanced proliferation and ECM synthesis, but suppressed apoptosis and inflammatory response in IL-1β-challenged HCs. Besides, silencing circADAMTS6 caused similar effects of IL-1β stress on HCs. Mechanically, there was a direct interaction between miR-324-5p and circADAMTS6 or PIK3R3, and IL-1β-induced activation of PI3K/AKT/mTOR signaling pathway was suppressed by circADAMTS6 overexpression and miR-324-5p silencing. Furthermore, counteractive effects of miR-324-5p upregulation on circADAMTS6 overexpression and PIK3R3 knockdown on miR-324-5p silencing were observed. CONCLUSION: CircADAMTS6-miR-324-5p-PIK3R3 axis might participate in IL-1β-induced HCs dysfunction via competing endogenous RNA mechanism and the PI3K/AKT/mTOR signaling pathway.